Despite decades of control effort, malaria remains rampant as a major global health problem, largely because of the emergence of drug resistance in the parasite and insecticide resistance in its mosquito vector. The search for alternate solutions is being pursued by rigorous research. Understanding of how parasite genes function and their products interact with their host is critical for understanding the transmission mechanism of this important human parasite. Our proposed research has two goals: (1) to achieve targeted gene replacement in the human malaria parasite Plasmodium falciparum; and (2) to perform gene function analysis in transgenic P. falciparum knockouts. As a model system, the P. falciparum Pfs25 gene will be targeted for gene knockout. This gene was chosen because of its potential importance in transmission of the sexual stages. In addition, because of its tight stage-specific expression, it is unlikely to be an essential gene for asexual parasite replication. Thus, either modification or replacement of this gene should be technically feasible in the asexual stages. We will design two types of vectors to target the Pfs25 gene to achieve gene replacement. One vector will mediate targeted gene replacement by double reciprocal recombination, the other by the "Hit and Run" pathway. Positive and negative selections will be used to enrich for homologous recombination between the cloned DNA and its corresponding sequence in the chromosome. As a long-term goal, we will initiate studies of parasite-vector interactions by introducing the transgenic knockouts into the mosquitoes to assess the biological consequences caused by the genetic manipulation.